Regulation of the immune response to both self-antigens and foreign pathogens is integral to host survival. The natural regulatory pathways involved may also afford opportunity to intercede in undesirable immune responses such as those occurring to allogeneic transplants. The attractiveness of this approach is evident in the enthusiasm for applying regulatory T cells (Tregs) to promote transplant survival in patients. Recent evidence suggests that parallel regulatory pathways may exist in the humoral arm of the immune response though the cells involved and the mechanisms of regulation are yet to be fully characterized. Despite the convincing evidence for regulatory B cells (Bregs) in non-transplant settings (autoimmunity, infectious disease, and bone marrow transplant/GVHD), a clear role for B cells or Bregs in the maintenance of induced transplant tolerance is lacking. During the last funding period we made the unexpected observation that a well-studied model of antibody induced transplantation tolerance depends on the presence of B lymphocytes and that graft prolongation in the model was transferrable to secondary hosts by B cells, indicating regulatory B cell activity. In the proposed studies, we will characterize the contribution of B cells to transplantation tolerance using models developed in the previous funding period. We will examine the hypothesis that B cells exert immunoregulatory action through cognate antigen presentation to donor strain-specific T cells and that this interaction impedes T cell immunity. We will also assess the contribution of the T cell Immunoglobulin and Mucin domain containing molecules (TIM-1 and TIM-4) to B cell-dependent tolerance and will apply a novel set of transgenic lines deficient in TIM-1 and TIM-4 expression to probe the possibility that TIM-1 and its ligand TIM-4 mark critical pathways in Breg-mediated tolerance. Finally, we will take advantage of our finding that anti-CD45RB-induced tolerance is dependent on both Tregs and B cells to explore the possibility that Treg development is fostered by Bregs or that Tregs and Bregs collaborate to promote graft acceptance. Collectively, these studies will further our understanding of the unique contribution of B cells to transplantation tolerance.